Smartphone-assisted point-of-care testing of nucleic acids based on hybridization chain reaction, magnetic beads, and gold nanorods etching

Colorimetric biosensors are simple but effective tools that are gaining popularity due to their ability to provide low-cost, rapid, and accurate detection for viruses like the Novel coronavirus, Influenza A, and Dengue virus, especially in point-of-care testing (POCT) and visual detection. In this study, a smartphone-assisted nucleic acid POCT was built using hybridization chain reaction (HCR), magnetic beads (MBs), and oxidized 3,3′,5,5′-tetramethylbenzidine (TMB2+)-mediated etching of gold nanorods (GNRs). The application of HCR without enzyme isothermal characteristics and MBs with easy separation, can quickly amplify nucleic acid signal and remove other reaction components. The blue shift of longitudinal localized surface plasmon resonance (LSPR) based on GNRs showed significant differences in etching color for different concentrations of target nucleic acid, which convert the signal into a visually semi-quantitative colorimetric result, achieving quantitative analysis with the color recognition software built into smartphones. This strategy, which only takes 40 min to detect and is two-thirds less time than the PCR, was successfully applied for the detection of the Dengue target sequence with a detection limit of 1.25 nM and exhibited excellent specificity for distinguishing single-base mutations, indicating broad application prospects in clinical laboratory diagnosis and enriching the research of nucleic acid POCT.

Droplet magnetic-controlled microfluidic chip integrated nucleic acid extraction and amplification for the detection of pathogens and tumor mutation sites.

Traditional nucleic acid extraction and detection is based on open operation, which may cause cross-contamination and aerosol formation. This study developed a droplet magnetic-controlled microfluidic chip integrated nucleic acid extraction, purification and amplification. The reagent is sealed in oil to form a droplet, and the nucleic acid is extracted and purified by controlling the movement of the magnetic beads (MBs) through a permanent magnet, ensuring a closed environment. This chip can automatically extract nucleic acid from multiple samples within 20 min, and can be directly placed in the in situ amplification instrument for amplification without further transfer of nucleic acid, characterized by simple, fast, time-saving and labor-saving. The results showed that the chip was able to detect <10 copies/test SARS-CoV-2 RNA, and EGFR exon 21 L858R mutations were detected in H1975 cells as low as 4 cells. In addition, on the basis of the droplet magnetic-controlled microfluidic chip, we further developed a multi-target detection chip, which used MBs to divide the nucleic acid of the sample into three parts. And the macrolides resistance mutations A2063G and A2064G, and the P1 gene of mycoplasma pneumoniae (MP) were successfully detected in clinical samples by the multi-target detection chip, providing the possibility for future application in the detection of multiple pathogens.

3d Printing of Magnetic Separator: An Affordable Approach to Sample Preparation in the Covid-19 Diagnosis

3D PRINTING OF MAGNETIC SEPARATOR: AN AFFORDABLE APPROACH TO SAMPLE PREPARATION IN THE COVID-19 DIAGNOSIS. This report describes the fabrication of a low-cost magnetic separator holder combining 3D printing and compact neodymium blocks for allowing magnetic extraction and purification of RNA from samples collected by nasopharyngeal swab from patients infected by SARS-CoV-2. The device was designed to contain 24 entrances for plastic microtubes in an arrangement like a commercial device. The proof of concept of the proposed device was successfully demonstrated through the sample extraction and purification of swab samples collected from eight patients suspected of SARS-CoV-2 infection. The sample preparation protocol was performed using a commercial kit containing magnetic beads and different solutions. The performance of the printed device was compared to a commercial magnetic separator, usually employed in the golden standard techniques. The fabrication of the 3D printed magnetic separator was completed under optimized printing conditions within 6 h at cost of 4 USD per unit. The RNA extracted from samples using both devices was analyzed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and the achieved results have indicated no statistical different at confidence level of 95%. Based on the achievements, the use of 3D printing and neodymium blocks have demonstrated an alternative route to be used in routing analysis associated to COVID-19 diagnosis.

Forty Years of Molecular Diagnostics for Infectious Diseases.

Nearly 40 years have elapsed since the invention of the PCR, with its extremely sensitive and specific ability to detect nucleic acids via in vitro enzyme-mediated amplification. In turn, more than 2 years have passed since the onset of the coronavirus disease 2019 (COVID-19) pandemic, during which time molecular diagnostics for infectious diseases have assumed a larger global role than ever before. In this context, we review broadly the progression of molecular techniques in clinical microbiology, to their current prominence. Notably, these methods now entail both the detection and quantification of microbial nucleic acids, along with their sequence-based characterization. Overall, we seek to provide a combined perspective on the techniques themselves, as well as how they have come to shape health care at the intersection of technologic innovation, pathophysiologic knowledge, clinical/laboratory logistics, and even financial/regulatory factors.

Solvent-Responsive Magnetic Beads for Accurate Detection of SARS-CoV-2.

Although numerous approaches were proposed for the nucleic acid (NA)-based SARS-CoV-2 detection, the nonideal NA desorption efficiency of conventional magnetic beads (MBs) limits their widespread application. In this study, we developed solvent-responsive MBs (called responsive MBs), which, in the presence of buffers, modulated the absorption and desorption capacities of NA by flipping the surface -COO-. Relative to other commercial MBs, responsive MBs exhibited similar absorption profiles and markedly enhanced desorption profiles. When applied for NA detection of complex samples, responsive MBs exhibited better performance of RNA detection than DNA, with obvious advantages in sensitivity. Specifically, the RNA and DNA desorption rates of commercial MBs were ∼85 and 82.5%, while those of responsive MBs were nearly 94 and 93.5%, respectively. Furthermore, responsive MBs exhibited remarkable extraction ability in a wide range of tissues and better performance of RNA extraction than DNA. When applied for SARS-CoV-2 detection, the responsive MBs along with the simulated digital RT-LAMP (a previously established apparatus) further improved detection efficiency, yielding a precise quantitative detection as low as 25 copies and an ultimate sensibility detection of 5 copies/mL. It was also successfully employed in numerous NA-based technologies such as polymerase chain reaction (PCR), sequencing, and so on.

Discovery and translation of functional nucleic acids for clinically diagnosing infectious diseases: Opportunities and challenges

Functional nucleic acids (FNAs) are short, single-stranded nucleic acids that can be derived from synthetic nucleic acid libraries using test-tube selection experiments. Due to their excellent chemical stability, high binding affinities and specificities, compatibility with a variety of signal-transduction mechanisms, and ease of synthesis and modification, FNAs have a great potential to overcome some of the limitations of current pathogen diagnostic methods by acting as molecular recognition elements (MREs) for point-of-care testing. This review summarizes the development of FNA-based biosensors for viral and bacterial detection in clinical samples. We first discuss examples of selecting FNAs for recognizing biomarkers of viral and bacterial pathogens. This is followed by discussion on integrating FNAs into fluorescent, colorimetric, and electrochemical biosensors and applying these sensors towards clinically diagnosing infectious diseases caused by many important bacterial and viral pathogens. Finally, the challenges of making FNA-based biosensors for infectious diseases are provided. (c) 2022 Elsevier B.V. All rights reserved.

Development and Validation of ScriptTaq COVID PCR: An In-House Multiplex rRT-PCR for Low-Cost Detection.

The COVID-19 pandemic necessitated an extensive testing for active SARS-CoV-2 infection. However, securing affordable diagnostic tests is a struggle for low-resource settings. We report herein the development and validation of an in-house multiplex real-time RT-PCR diagnostic test for the detection of active COVID-19 infection (ScriptTaq COVID PCR). Furthermore, we describe two methods for RNA extraction using either an in-house silica column or silica-coated magnetic beads to replace commercial RNA extraction kits. Different buffer formulations for silica column and silica-coated magnetic beads were tested and used for RNA isolation. Taq polymerase enzyme and thermostable reverse transcriptase enzyme were purified from bacterial clones. Primers/probes sequences published by the WHO and CDC were used for the qualitative detection of the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes, respectively. ScriptTaq COVID PCR assay was able to detect up to 100 copies per reaction of the viral RdRP and N genes. The test demonstrated an overall agreement of 95.4%, a positive percent agreement (PPA) of 90.2%, and a negative percent agreement (NPA) of 100.0% when compared with two commercially available kits. ScriptTaq COVID PCR diagnostic test is a specific, sensitive, and low-cost alternative for low-resource settings.

Rapid Extraction of Viral Nucleic Acids Using Rotating Blade Lysis and Magnetic Beads.

The complex and lengthy protocol of current viral nucleic acid extraction processes limits their use outside laboratory settings. Here, we describe a rapid and reliable method for extracting nucleic acids from viral samples using a rotating blade and magnetic beads. The viral membrane can be instantly lysed using a high-speed rotating blade, and nucleic acids can be immediately isolated using a silica magnetic surface. The process was completed within 60 s by this method. Routine washing and eluting processes were subsequently conducted within 5 min. The results achieved by this method were comparable to those of a commercially available method. When the blade-based lysis and magnetic bead adsorption processes were performed separately, the RNA recovery rate was very low, and the Ct value was delayed compared to simultaneous lysis and RNA adsorption. Overall, this method not only dramatically shortens the conventional extraction time but also allows for its convenient use outside the laboratory, such as at remote field sites and for point-of-care testing.

A magnetic bead immunoassay to detect high affinity human IgG reactive to SARS-CoV-2 Spike S1 RBD produced in Escherichia coli.

Immunological assays to detect SARS-CoV-2 Spike Receptor Binding Domain (RBD) antigen seroconversion in humans are important tools to monitor the levels of protecting antibodies in the population in response to infection and/or immunization. Here we describe a simple, low cost, and high throughput Ni2+ magnetic bead immunoassay to detect human IgG reactive to Spike S1 RBD Receptor Binding Domain produced in Escherichia coli. A 6xHis-tagged Spike S1 RBD was expressed in E. coli and purified by affinity chromatography. The protein was mobilized on the surface of Ni2+ magnetic beads and used to investigate the presence of reactive IgG in the serum obtained from pre-pandemic and COVID-19 confirmed cases. The method was validated with a cohort of 290 samples and an area under the receiver operating characteristic curve of 0.94 was obtained. The method was operated with > 82% sensitivity at 98% specificity and was also able to track human IgG raised in response to vaccination with Comirnaty at > 85% sensitivity. The IgG signal obtained with the described method was well-correlated with the signal obtained when pre fusion Spike produced in HEK cell lines was used as antigen. This novel low-cost and high throughput immunoassay may act as an important tool to investigate protecting IgG antibodies against SARS-CoV-2 in the human population.

SAMPLE-TO-ANSWER WHOLE BLOOD SEROLOGICAL TEST FOR SARS-COV-2 USING THERMALLY RESPONSIVE VALVES

To maximize access to SARS-CoV-2 serological (antibody) tests, point-of-care (PoC) options are necessary. PoC tests require sample-to-answer functionality, which is challenging with whole blood. Here we demonstrate a sample-to-answer SARS-CoV-2 antibody test from whole blood using automated thermally actuated valves. Higher-order alkanes serve as partitions between immunoassay zones (sample/bind, rinse, detection);upon warming, the partitions are liquified, enabling magnetic beads to be pulled through each zone while continuing to partition the reagents. We demonstrate a detection limit of 0.7 ng/mL SARS-CoV-2 antibodies, multiple orders of magnitude lower than clinically relevant concentrations. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Useful molecular tools for facing next pandemic events: Effective sample preparation and improved RT-PCR for highly sensitive detection of SARS-CoV-2 in wastewater environment.

Viral pandemics can be inevitable in the next future. Considering SARS-CoV-2 pandemics as an example, there seems to be a need to develop a surveillance system able to monitor the presence of potential pathogenic agents. The sewage and wastewater environments demonstrated to be suitable targets for such kind of analysis. In addition, it is important to have reliable molecular diagnostic tools and also to develop a robust detection strategy. In this study, an effective sample preparation procedure was selected from four options and combined with a newly developed improved RT-PCR. First, a model viral system was constructed, containing a fragment of the SARS-CoV-2 gene encoding for the Spike protein. The encapsidated S RNA mimic (ESRM) was based on the plum pox virus (PPV) genome with the inserted targeted gene fragment. ESRM was used for seeding wastewater samples in order to evaluate the viral recovery of four different viral RNA concentration/extraction methods. The efficiency of individual approaches was assessed by the use of a quantitative reverse transcription PCR (qRT-PCR) and by a one-step single-tube nested quantitative reverse transcription PCR (OSN-qRT-PCR). For the detection of viruses in wastewater samples with low viral loads, OSN-qRT-PCR assay produced the most satisfactory results and the highest sensitivity.

Identification of inhibitors for the S1-ACE2 interaction of the SARS-CoV-2 using magnetically modulated biosensors

The outbreak of the coronavirus disease emphasized the need for fast and sensitive inhibitor screening tools for the identification of new drug candidates. In SARS-CoV-2, one of the initial steps in the infection cycle is the adherence of the receptor-binding domain (RBD) of the spike protein 1 (S1) to the host cell by binding to the angiotensin-converting enzyme 2 (ACE2) receptor. Therefore, inhibition of S1-ACE2 interaction may block the entry of the virus to the host cell, and thus may limit the spread of the virus in the body. We demonstrate a rapid and quantitative method for the detection and classification of different types of molecules as inhibitors or non-inhibitors of the S1-ACE2 interaction using magnetically modulated biosensors (MMB). In the MMB-based assay, magnetic beads are attached to the S1 protein and the ACE2 receptor is fluorescently labeled. Thus, only when the proteins interact, the fluorescent molecule is connected to the magnetic bead. To increase the sensitivity of fluorescence detection, the complex of magnetic beads and attached fluorescent molecules are aggregated by two opposing electromagnets and are moved from side to side in a periodic motion in and out of a laser beam, emitting a flashing signal that is collected by a digital camera. When an inhibitor interferes with the interaction, the signal is reduced. The MMB-based assay is much faster and has minimal non-specific binding than the commonly used ELISA. It can be adjusted to other interactions, and therefore can be utilized as a global tool for inhibitor screening. © COPYRIGHT SPIE. Downloading of the is permitted for personal use only.

High throughput optical modulation biosensing for highly sensitive and rapid detection of biomarkers.

Rapid, highly sensitive, and high-throughput detection of biomarkers at low concentrations is invaluable for early diagnosis of various diseases. In many highly sensitive immunoassays, magnetic beads are used to capture fluorescently labeled target molecules. The target molecules are then quantified by detecting the fluorescent signal from individual beads, which is time consuming and requires a complicated and expensive detection system. Here, we demonstrate a high-throughput optical modulation biosensing (ht-OMB) system, which uses a small permanent magnet to aggregate the beads into a small detection volume and eliminates background noise by steering a laser beam in and out of the cluster of beads. Shortening the aggregation, acquisition, and well-to-well scanning transition times enables reading a 96-well plate within 10 min. Using the ht-OMB system to detect human Interleukin-8, we demonstrated a limit of detection of 0.14 ng/L and a 4-log dynamic range. Testing 94 RNA extracts from 36 confirmed RT-qPCR SARS-CoV-2-positive patients (Ct≤40) and 58 confirmed RT-qPCR SARS-CoV-2-negative individuals resulted in 100% sensitivity and 100% specificity.

SARS-CoV-2 electrochemical immunosensor based on the spike-ACE2 complex.

Rapid, straightforward, and massive diagnosis of coronavirus disease 2019 (COVID-19) is one of the more important measures to mitigate the current pandemics. This work reports on an immunosensor to rapidly detect the spike protein from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The immunosensing device entraps the spike protein linked to angiotensin-converting enzyme host receptor (ACE2) protein in a sandwich between carboxylated magnetic beads functionalized with an anti-spike antibody and an anti-ACE2 antibody, further labeled with streptavidin (poly)horseradish peroxidase (HRP) reporter enzyme. The particles were confined at the surface of screen-printed gold electrodes, whose signal resulting from the interaction of the enzyme with a mediator was recorded in a portable potentiostat. The immunosensor showed a sensitivity of 0.83 µA∗mL/µg and a limit of detection of 22.5 ng/mL of spike protein, with high reproducibility. As a proof-of-concept, it detected commercial spike protein-supplemented buffer solutions, pseudovirions, isolated viral particles and ten nasopharyngeal swab samples from infected patients compared to samples from three healthy individuals paving the way to detect the virus closer to the patient.

Voltammetric biosensor for coronavirus spike protein using magnetic bead and screen-printed electrode for point-of-care diagnostics.

The rapid spread of the novel human coronavirus 2019 (COVID-19) and its morbidity have created an urgent need for rapid and sensitive diagnostics. The real-time polymerase chain reaction is the gold standard for detecting the coronavirus in various types of biological specimens. However, this technique is time consuming, labor intensive, and expensive. Screen-printed electrodes (SPEs) can be used as point-of-care devices because of their low cost, sensitivity, selectivity, and ability to be miniaturized. The ability to detect the spike protein of COVID-19 in serum, urine, and saliva was developed using SPE aided by magnetic beads (MBs) and a portable potentiostat. The antibody-peroxidase-loaded MBs were the captured and catalytic units for the electrochemical assays. The MBs enable simple washing and homogenous deposition on the working electrode using a magnet. The assembly of the immunological MBs and the electrochemical system increases the measuring sensitivity and speed. The physical and electrochemical properties of the layer-by-layer modified MBs were systematically characterized. The performance of these immunosensors was evaluated using spike protein in the range 3.12-200 ng mL-1. We achieved a limit of detection of 0.20, 0.31, and 0.54 ng mL-1 in human saliva, urine, and serum, respectively. A facile electrochemical method to detect COVID-19 spike protein was developed for quick point-of-care testing.

A general controllable release amplification strategy of liposomes for single-particle collision electrochemical biosensing.

As an important component of the COVID-19 mRNA vaccines, liposomes play a key role in the efficient protection and delivery of mRNA to cells. Herein, due to the controllable release amplification strategy of liposomes, a reliable and robust single-particle collision electrochemical (SPCE) biosensor was constructed for H9N2 avian influenza virus (H9N2 AIV) detection by combining liposome encapsulation-release strategy with immunomagnetic separation. The liposomes modified with biotin and loaded with platinum nanoparticles (Pt NPs) were used as signal probes for the first time. Biotin facilitated the coupling of biomolecules (DNA or antibodies) through the specific reaction of biotin-streptavidin. Each liposome can encapsulate multiple Pt NPs, which were ruptured under the presence of 1 × PBST (phosphate buffer saline with 0.05% Tween-20) within 2 min, and the encapsulated Pt NPs were released for SPCE experiment. The combination of immunomagnetic separation not only improved the anti-interference capabilities but also avoided the agglomeration of Pt NPs, enabling the SPCE biosensor to realize ultrasensitive detection of 18.1 fg/mL H9N2 AIV. Furthermore, the reliable SPCE biosensor was successfully applied in specific detection of H9N2 AIV in complex samples (chicken serum, chicken liver and chicken lung), which promoted the universality of SPCE biosensor and its application prospect in early diagnosis of diseases.

Development of Viral Genome Extraction Machine

The world has been hit by coronavirus pandemic for around two years. Early detection of infection by SARS CoV-2 relies on the efficient detection using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) which require a viral genome extraction machine. An extraction machine for the nucleic acid has been designed and fabricated in this research. It utilizes magnetic rods and carousel driven by linear and rotary actuator as the main component to do each step of extraction procedure. It is equipped with a minicomputer and Liquid Crystal Display (LCD) touchscreen as an interface with user to make setting and running the machine. The machine has been tested to simulate each extraction process without viral genome sample comprises lysis, two times washing, holding and elution as designed. It was running well to rotate the carousel to the exact position for each extraction step, move the tip comb and magnetic rods appropriately to the sample plate holes, and move the tip comb up and down to mix the solution exactly on the sample plate for each extraction process. Next, the machine performance will be tested to do viral genome extraction in BSL Laboratory Class 2. © 2021 IEEE.

A novel approach to concentrate human and animal viruses from wastewater using receptors-conjugated magnetic beads.

Viruses are present at low concentrations in wastewater; therefore, an effective method for concentrating virus particles is necessary for accurate wastewater-based epidemiology (WBE). We designed a novel approach to concentrate human and animal viruses from wastewater using porcine gastric mucin-conjugated magnetic beads (PGM-MBs). We systematically evaluated the performances of the PGM-MBs method (sensitivity, specificity, and robustness to environmental inhibitors) with six viral species, including Tulane virus (a surrogate for human norovirus), rotavirus, adenovirus, porcine coronavirus (transmissible gastroenteritis virus or TGEV), and two human coronaviruses (NL63 and SARS-CoV-2) in influent wastewater and raw sewage samples. We determined the multiplication factor (the ratio of genome concentration of the final solution to that of the initial solution) for the PGM-MBs method, which ranged from 1.3 to 64.0 depending on the viral species. Because the recovery efficiency was significantly higher when calculated with virus titers than it was with genome concentration, the PGM-MBs method could be an appropriate tool for assessing the risk to humans who are inadvertently exposed to wastewater contaminated with infectious viruses. Furthermore, PCR inhibitors were not concentrated by PGM-MBs, suggesting that this tool will be successful for use with environmental samples. In addition, the PGM-MBs method is cost-effective (0.5 USD/sample) and has a fast turnaround time (3 h from virus concentration to genome quantification). Thus, this method can be implemented in high throughput facilities. Because of its strong performance, intrinsic characteristics of targeting the infectious virus, robustness to wastewater, and adaptability to high throughput systems, the PGM-MBs method can be successfully applied to WBE and ultimately provides valuable public health information.

Reversible superhydrophobicity unyielding magnetic beads of flipping-triggered (SYMBOL) regulate the binding and unbinding of nucleic acids for ultra-sensitive detection

Present molecular biology procedures often include extraction and purification of nucleic acids (NAs) based on centrifuge- and column-based methods, which involve the usage of specialized apparatus and toxic chemicals. These restrict its usage in large scale high-throughput settings. Magnetic beads (MBs) may provide an ideal approach to extracting and purifying DNA or RNA instead of them. However, the low separation rate of NAs (DNA or RNA) on MBs limits its NAs’ detection. Here, we developed a reversible superhydrophobicity unyielding magnetic-beads of flipping-triggered (SYMBOL) system which can regulate the binding and unbinding capacity of NAs via the buffers due to the flipping of –COO- on the surface of SYMBOL. Compared to other MBs examined in this study, SYMBOL exhibited similar binding properties and far enhanced separation properties. In fact, the DNA separation rate rose from about 79% to nearly 96%, whereas, the RNA separation rate increased from roughly 75% to 97%. When it applied to SARS-CoV-2 detection, the SYMBOL, coupled with Simulation Digital RT-LAMP (a previously developed system), could further increase the sensitivity of the detection, thereby achieving a detection threshold of 5 copies/mL. The SYMBOL showed excellent extraction capability in a variety of tissues and can be used in a wide range of nucleic acid related technologies such as PCR, sequencing, etc. © 2021 The Author(s)

Ultra-fast, high throughput and inexpensive detection of SARS-CoV-2 seroconversion using Ni2+ magnetic beads.

To monitor the levels of protecting antibodies raised in the population in response to infection and/or to immunization with SARS-CoV-2, we need a technique that allows high throughput and low-cost quantitative analysis of human IgG antibodies reactive against viral antigens. Here we describe an ultra-fast, high throughput and inexpensive assay to detect SARS-CoV-2 seroconversion in humans. The assay is based on Ni2+ magnetic particles coated with His tagged SARS-CoV-2 antigens. A simple and inexpensive 96 well plate magnetic extraction/homogenization process is described which allows the simultaneous analysis of 96 samples and delivers results in 7 min with high accuracy.